The group of soluble secreted molecules, collectively termed cytokines, represents critical communication signals among the cells of the immune system and between immune and non-immune system cells. Some of these cytokines have to form homodimers, such as interferon-gamma (IFN-γ), or homotrimers, such as tumor necrosis factor (TNF)-family molecules, in order to exert biological activity. Monomeric forms show minimal or no bioactivity.
Cytokines are at present used in drugs in cancer therapy and in the combat against chronic microbial infections. Cytokines are also evaluated for their use as immune stimulators in adjuvants to improve vaccines.
In mammals, a group of composite hetero-dimeric cytokines has been identified based on complexes of a p40 protein subunit. The mammalian p40 element comprises a 40 kD protein which links covalently, by di-sulfide binding, with a p35 subunit to form interleukin-12 (IL-12) p70 (Gubler U, et al., PNAS USA 1991, 88: 4143-4147; Wolf S F, et al., J. Immunol. 1991, 146: 3047-3081; Trinchieri G., Blood 1994, 12: 4008-4027). In addition, p40 may form the composite cytokine IL-23, after combining with p19 (Wiekowski M T, et al., J. Immunol. 2001 166: 7563-7570), a molecule structurally related to IL-6, p35 and granulocyte-colony stimulating factor (G-CSF) (Oppmann B, et al., Immunity 2000, 13: 715-725). Moreover, p40 may form homodimers that have been shown to either compete for binding with IL-12 p70 to the IL-12 high affinity receptor and inhibit IL-12 bioactivity (Heinzel F P, et al., J. Immunol., 1997, 158: 4381-4388), or to enhance, rather than to decrease, IFN-γ production by CD8+ T cells and Th1 development (Piccotti J R, et al., J. Immunol. 1997, 158: 643-648).
The significance of p40 in vivo in various mammalian species has been demonstrated using recombinant p40. IL-12 antagonistic features of 80 KDa homodimeric (P40)2 have been clearly demonstrated in lipopolysaccharide (LPS)-induced IFN-γ-dependent lethal shock models (Mattner F, et al., Infect. Immun. 1997, 11: 4734-4737). The production of human p40, in the absence of bioactive IL-12 p70, has been demonstrated for brain microglial cells (De Goer-de Herve M G, et al., Cytokine 2001, 14: 88-96). In addition, the physiological role of mammalian p40 composite cytokines has been delineated in detail using in vivo gene-targeting approaches. It proved that following infection by Salmonella enteritidis mice genetically deficient for the p40 protein (p40−/−) showed a higher mortality rate and higher bacterial organ burden than mice capable of producing p40, but lacking the p35 gene (IL-12 p35−/−) (Lehmann J, et al., J. Immunol. 2001, 167: 5304-5315). Normal (wild-type) and IL-12 p35−/− mice cleared an infection with Mycobacterium bovis Calmette-Guerin (BCG) or pulmonary tuberculosis infection, while double-deficient IL-12 (p35−/−+p40−/−) mice showed high susceptibility to M. bovis BCG and tuberculosis infection (Holscher C, et al., J. Immunol. 2001, 167: 6957-6566). Susceptibility was associated with reduced antigen-specific Th1 and cytotoxic T cell responses. Interestingly, in vivo therapy with recombinant p40 homodimers reverted M. bovis BCG infected double-depleted (p35−/−+p40−/−) mice into a resistant phenotype. This demonstrates a protective and agonistic role of endogenous and exogenous p40 in mycobacterial infection, which is independent of IL-12 p70 (Holscher et al., 2001 supra). Similarly, Cryptococcus neoformans infected p40−/− mice died earlier and developed higher organ burdens than p35−/− mice, which suggests again a protective role for the p40 subunit independent of the IL-12 heterodimer (Decken K., et al., Infect. Immunity 1998, 66: 4994-5000). Also, p40−/− mice survived large doses of the intracellular bacterium Franscisella tularensis (LVS), but never cleared bacteria and developed chronic infection. In sharp contrast, p35−/− mice readily survived large doses of sub lethal LVS infection. This study suggests that clearance of LVS is dependent on p40 but not on IL-12 p70 (Elkins K L, et al., Infect. Immun. 2002, 70: 1936-1946). Also during murine cytomegalovirus (MCMV) infection p35−/− mice showed an altered phenotype compared to p40−/− mice, indicating that p40 may have an activity independent of and additional to IL-12 antagonism in vivo (Carr J A, et al., J. Interferon Cytokine Res. 1999, 19: 1145-1152).
Taken together, these experimental studies illustrate the crucial role in mammals of p40 based cytokines such as IL-12, IL-23 and (p40)2 in regulation of IFN-γ characteristic T helper-1 type immune responses essential in the control of mostly intracellular infections of bacterial, parasitic, fungal or viral nature.
The present invention concerns avian equivalents of the mammalian p40 based cytokines.
The cloning and sequencing of avian cytokines lags behind similar work done in mammals. Only a few avian cytokines have been identified so far. IFN-γ and IL-18 as well as a number of pro-inflammatory cytokines have been cloned, demonstrating the existence of a Th1-like cytokine network in chickens. Because of the low sequence homology to mammalian cytokines, usually somewhere around 30 to 50%, classical approaches to identify avian homologues of mammalian cytokines are usually not successful. The identification by PCR amplification using primers based on mammalian sequences is very difficult and unpredictive. (Hilton L. S. et al. Vet. Immunol. and Immunopathol. 2002, 85: 119-128; Staehi P. et al., J. Interferon Cytokine Res. 2001, 21: 993-1010) When some avian cytokines became available, work started to investigate their potential use as immune modulators or as immune adjuvants to enhance the efficiency of vaccines.
Most chickens produced in developed countries, both for consumption and egg-laying, are vaccinated. They are vaccinated against Marek's disease, and against Newcastle Disease Virus, Infectious Bursal Disease Virus, Infectious Bronchitis Virus, Fowlpox Virus, and Coccidial vaccines. Vaccination can be performed either before or after hatching. The immune systems of embryos and newly hatched birds is not yet fully developed and cannot give rise to an immune response that is as effective as 2-3 weeks after hatching. For the development of vaccines used pre-hatching or at-hatching, therefore a need exists for agents that enhance the immune response in birds after vaccination.
The present inventors have succeeded in identifying and determining both the amino acid- and the encoding gene sequence for novel avian cytokines. These proteins are useful for the above-mentioned purposes known for the mammalian counterparts, especially to enhance the effectiveness of avian vaccines.